The long term objective of the proposed research is to delineate at the molecular level the structural determinants responsible for the formation, regulation, and mechanism of action of the two key enzymes of complement activation, the classical and alternative pathway C3 convertases. We are particularly interested in describing critical details on the topology, structure, and chemical nature of recognition and effector sites of factor D, factor B, and C2. We propose to approach our objective by pursuing three specific aims: 1) Define structural correlates of factor D function. We will utilize newly obtained information on the x-ray crystal structure of factor D to construct mutants aimed at describing residues important for substrate binding and catalysis. Oligonucleotide-directed site- specific mutagenesis and expression in eukaryotic systems will be utilized in these experiments. 2) Define the site and mechanism of activation of proD. These experiments will be based on our recent data on proD expression and activation. The subcellular localization of proD will be investigated by using monoclonal antibodies and efforts will be made to identify the proD processing enzyme. 3) Describe C4b- and C3b-binding sites on C2 and factor B, respectively. These studies will use site- directed and deletional mutagenesis to construct mutants and C2/factor B hybrids that will allow for the definition of protein binding sites. Recently isolated alternatively-spliced C2 gene transcripts encoding truncated C2 proteins will also be used in these experiments. Complement-mediated acute inflammation leads to host tissue injury in a variety of human diseases. The data we propose to collect are necessary for attaining the medically important goal of pharmacologic control of complement activation in these diseases.